Dataset Information

Development of Unlabeled Probe Based High-Resolution Melting Analysis for Detection of Filaggrin Gene Mutation c.3321delA.

ABSTRACT: BACKGROUND:Filaggrin gene (FLG) plays an important role in skin barrier function, and loss-of-function mutations of FLG have been shown to be a predisposing factor for atopic dermatitis (AD). The c.3321delA mutation is the most common FLG mutation in Chinese population. We aim to develop a rapid, cost-efficiency, and reliable closed-tube method that has not been described for the detection of c.3321delA mutation. METHODS:Recombinant wild-type and mutant plasmids of c.3321delA mutation were constructed, heterozygous mutant plasmids were prepared by mixing the mutant plasmids and wild-type plasmids at 1:1 ratio. High-resolution melting analysis (HRMA) coupled with an unlabeled DNA probe was employed to identify the shift in melting temperature of the probe-template complex, which reflects the presence of c.3321delA mutation. RESULTS:Unlabeled probe based HRMA was able to distinguish all three genotypes (wild-type, heterozygote, and mutant) of c.3321delA mutation. Then, we applied this method to genotype 1,317 clinical samples. Genotyping results obtained from unlabeled probe HRMA were 100% concordant with the results from direct sequencing. CONCLUSION:We developed a fast and high-throughput method to detect the c.3321delA mutation.

SUBMITTER: Zhong WL

PROVIDER: S-EPMC6806714 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Development of Unlabeled Probe Based High-Resolution Melting Analysis for Detection of Filaggrin Gene Mutation c.3321delA.

Zhong Wei-Long WL Wang Luo L Wu Xia X Zhang Jie J Chen Xiao-Fan XF Zhang Wei W Dou Xia X Yu Bo B

Journal of clinical laboratory analysis 20160403 6

<h4>Background</h4>Filaggrin gene (FLG) plays an important role in skin barrier function, and loss-of-function mutations of FLG have been shown to be a predisposing factor for atopic dermatitis (AD). The c.3321delA mutation is the most common FLG mutation in Chinese population. We aim to develop a rapid, cost-efficiency, and reliable closed-tube method that has not been described for the detection of c.3321delA mutation.<h4>Methods</h4>Recombinant wild-type and mutant plasmids of c.3321delA muta ...[more]

PMID: 27040325

Similar Datasets

Project description:With looming resistance to fluoroquinolones in Mycoplasma genitalium, public health control strategies require effective antimicrobial resistance (AMR) diagnostic methods for clinical and phenotypic AMR surveillance. We developed a novel AMR detection method, MGparC-AsyHRM, based on the combination of asymmetric high-resolution melting (HRM) technology and unlabeled probes, which simultaneously performs M. genitalium identification and genotypes eight mutations in the parC gene that are responsible for most cases of fluoroquinolone resistance. These enhancements expand the traditional HRM from the conventional detection of single-position mutations to a method capable of detecting short fragments with closely located AMR positions with a high diversity of mutations. Based on the results of clinical sample testing, this method produces an accordance of 98.7% with the Sanger sequencing method. Furthermore, the specificity for detecting S83I, S83N, S83R, and D87Y variants, the most frequently detected mutations in fluoroquinolone resistance, was 100%. This method maintained a stable and accurate performance for genomic copies at rates of ≥20 copies per reaction, demonstrating high sensitivity. Additionally, no specific cross-reactions were observed when testing eight common sexually transmitted infection (STI)-related agents. Notably, this work highlights the significant potential of our method in the field of AMR testing, with the results suggesting that our method can be applied in a range of scenarios and to additional pathogens. In summary, our method enables high throughput, provides excellent specificity and sensitivity, and is cost-effective, suggesting that this method can be used to rapidly monitor the molecular AMR status and complement current AMR surveillance. IMPORTANCE Mycoplasma genitalium was recently added to the antimicrobial-resistant (AMR) threats "watch list" of the U.S. Centers for Disease Control and Prevention because this pathogen has become extremely difficult to treat as a result of increased resistance. M. genitalium is also difficult to culture, and therefore, molecule detection is the only method available for AMR testing. In this work, we developed a novel AMR detection method, MGparC-AsyHRM, based on the combination of asymmetrical HRM technology and unlabeled probes, and it simultaneously performs M. genitalium identification and genotypes eight mutations in the parC gene that are responsible for most cases of fluoroquinolone resistance. The MGparC-AsyHRM method is a high-throughput, low-cost, simple, and culture-free procedure that can enhance public health and management of M. genitalium infections and AMR control, providing a strong complement to phenotypic AMR surveillance to address the spread of fluoroquinolone resistance.

Project description:BACKGROUND:Mutation scanning provides the simplest, lowest-cost method for identifying DNA variations on single PCR amplicons, and it may be performed before sequencing to avoid screening of noninformative wild-type samples. High-resolution melting (HRM) is the most commonly used method for mutation scanning. With PCR-HRM, however, mutations less abundant than approximately 3%-10% that can still be clinically significant may often be missed. Therefore, enhancing HRM detection sensitivity is important for mutation scanning and its clinical application. METHODS:We used serial dilution of cell lines containing the TP53 exon 8 mutation to demonstrate the improvement in detection sensitivity for conventional-PCR-HRM in the presence of DMSO. We also conducted coamplification at lower denaturation temperature (COLD)-PCR with an extra step for cross-hybridization, followed by preferential denaturation and amplification at optimized critical temperature (full-COLD-PCR), to further enrich low-level mutations before HRM with or without DMSO, and we used droplet-digital PCR to derive the optimal conditions for mutation enrichment. Both conventional PCR-HRM and full-COLD-PCR-HRM with and without DMSO were used for mutation scanning of TP53 exon 8 in cancer samples containing known mutations and myelodysplastic syndrome samples with unknown mutations. Mutations in other genes were also examined. RESULTS:The detection sensitivity of PCR-HRM scanning increases 2- to 5-fold in the presence of DMSO, depending on mutation type and sequence context, and can typically detect mutation abundance of approximately 1%. When mutation enrichment is applied during amplification with full-COLD-PCR followed by HRM in the presence of DMSO, mutations with 0.2%-0.3% abundance in TP53 exon 8 can be detected. CONCLUSIONS:DMSO improves HRM mutation scanning sensitivity with saturating dyes. When full-COLD-PCR is used, followed by DMSO-HRM, the overall improvement is about 20-fold compared with conventional PCR-HRM.

Project description:BACKGROUND: TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful tool for reverse genetics, combining traditional chemical mutagenesis with high-throughput PCR-based mutation detection to discover induced mutations that alter protein function. The most popular mutation detection method for TILLING is a mismatch cleavage assay using the endonuclease CelI. For this method, locus-specific PCR is essential. Most wheat genes are present as three similar sequences with high homology in exons and low homology in introns. Locus-specific primers can usually be designed in introns. However, it is sometimes difficult to design locus-specific PCR primers in a conserved region with high homology among the three homoeologous genes, or in a gene lacking introns, or if information on introns is not available. Here we describe a mutation detection method which combines High Resolution Melting (HRM) analysis of mixed PCR amplicons containing three homoeologous gene fragments and sequence analysis using Mutation Surveyor software, aimed at simultaneous detection of mutations in three homoeologous genes. RESULTS: We demonstrate that High Resolution Melting (HRM) analysis can be used in mutation scans in mixed PCR amplicons containing three homoeologous gene fragments. Combining HRM scanning with sequence analysis using Mutation Surveyor is sensitive enough to detect a single nucleotide mutation in the heterozygous state in a mixed PCR amplicon containing three homoeoloci. The method was tested and validated in an EMS (ethylmethane sulfonate)-treated wheat TILLING population, screening mutations in the carboxyl terminal domain of the Starch Synthase II (SSII) gene. Selected identified mutations of interest can be further analysed by cloning to confirm the mutation and determine the genomic origin of the mutation. CONCLUSION: Polyploidy is common in plants. Conserved regions of a gene often represent functional domains and have high sequence similarity between homoeologous loci. The method described here is a useful alternative to locus-specific based methods for screening mutations in conserved functional domains of homoeologous genes. This method can also be used for SNP (single nucleotide polymorphism) marker development and eco-TILLING in polyploid species.

Dataset Information

Development of Unlabeled Probe Based High-Resolution Melting Analysis for Detection of Filaggrin Gene Mutation c.3321delA.

Publications

Development of Unlabeled Probe Based High-Resolution Melting Analysis for Detection of Filaggrin Gene Mutation c.3321delA.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets